Molecular allergy diagnosis is sensitive and avoids misdiagnosis in patients sensitized to seasonal allergens

Abstract Background The specificity of extract‐based pollen allergy diagnosis is decreased due to cross‐reactivity via cross‐reactive carbohydrate determinants (CCDs) or panallergens such as profilins or polcalcins. This study aimed to explore the prevalence of sensitization to seasonal extracts, CCDs, profilin and polcalcin and investigate the sensitivity and specificity of seasonal molecular allergy diagnosis (MAD) using commercially available test methods. Methods 2948 patients were screened for specific immunoglobulin E to ash, birch, mugwort, ragweed and timothy grass pollen extracts and grouped according to the number of positive tests (1–5). 100 patients from each group and a control group were randomly selected to calculate the prevalence of CCD and panallergen sensitization. With 742 patients, sensitivity and specificity of MAD (Alt a 1, Fra/Ole e 1, Bet v 1, Phl p 1, Art v 1, and Amb a 1) was determined. Results 1627 patients (55.2%) were positive to at least one, and 1002 patients (34.0%) were positive to multiple of the five pollen allergens investigated; 18.5% of the pollen‐sensitized patients had sensitization to CCDs or panallergens. Specifically, sensitization to CCDs, profilins, and polcalcins was observed in 8.7%, 10.9%, and 2.9% of these patients, respectively. The sensitivity of MAD was high, with sensitivities between 96.2% and 100% using ImmunoCAP and 91.5% and 100% using ALEX2. Specificity was 100% for both assays. Conclusions Due to cross‐reactivity, about one‐fifth of pollen‐sensitized patients is at risk of misdiagnosis. However, MAD is sensitive, specific and helps to avoid misdiagnosis and select primary allergen sources for immunotherapy.


| INTRODUCTION
Allergic rhinoconjunctivitis with or without asthma is one of the most frequent allergic diseases, affecting about 20% of the general population. 1 Allergen immunotherapy is the only available treatment that targets the underlying pathophysiology and has a potential long-term effect on reducing allergic symptoms. Selecting the appropriate (major) allergens for immunotherapy is crucial to achieve optimal effectiveness. Currently, prick testing and extractbased specific immunoglobulin E (sIgE) determination are still the mainstays in diagnosing respiratory allergy. However, it has been known for years that cross-reactive carbohydrate determinants (CCDs) or panallergens such as profilins or polcalcins can hamper or confound test results with native extracts by decreasing diagnostic specificity.
This study had two goals. First, we aimed to determine the prevalence of multiple pollen sensitizations and of cross-reactivity (via sensitization to CCDs, profilin, and polcalcin) in patients referred to allergy diagnostics and in patients with pollen sensitization. Second, we evaluated the sensitivity and specificity of molecular allergy diagnosis (MAD) with major seasonal marker allergens using the commercially available singleplex platform ImmunoCAP (Thermo Fisher Scientific, Waltham, MA, USA) and the multiplex array ALEX 2 (Allergy Explorer version 2, MacroArray Diagnostics, Vienna, Austria).

| METHODS
For this retrospective study, clinical data and extract and prick test results of 3590 patients were investigated using four analyses ( Figure 1). None of the patients in any of the analyses currently or previously received allergen-specific immunotherapies.

| Analysis 1. Prevalence of pollen sensitization
The frequency of (multiple) pollen sensitization was investigated in 2948 patients with rhinoconjunctivitis and/or bronchial asthma referred for allergy diagnostics between January 1 and December 31, 2020. Subjects were screened for pollen allergy using the determination of sIgE against five pollen extracts (ash, birch, timothy grass, mugwort, and ragweed) by ImmunoCAP. We classified subjects according to their number of sensitizations; patients showing sIgE to one to five different pollen extracts were grouped accordingly. In addition, because Alternaria is another important seasonal allergen in our region, prevalence of sIgE sensitization to Alternaria was investigated in all 2948 patients.

| Analysis 2. Sensitization to CCDs and pollen panallergens
One hundred persons from each of the five groups mentioned above and a control group of 100 non-allergic subjects were randomly selected. All controls had negative sIgE to the investigated six seasonal allergen extracts and Dermatophagoides pteronyssinus in the ImmunoCAP system and negative prick tests to 13 aeroallergens (pollen, pets, mites and moulds). In these 600 sera, the markers MUXF3 (ImmunoCAP), Phl p 7 (ALEX 2 ), and Phl p 12 (ALEX 2 ) were determined to detect the frequency of sensitization to CCDs, profilin, and polcalcin, respectively.

| Analysis 3. Sensitivity of major marker allergens
To calculate the sensitivity of the molecular marker allergens of these five pollen species (Fra/Ole e 1, Bet v 1, Phl p 1, Art v 1, and Amb a 1) and Alternaria (Alt a 1), 636 patients visiting the outpatient clinic between 2013 and 2020 were included. All these patients had positive sIgE and prick tests to the respective extract and reported seasonal respiratory symptoms limited to the pollination period of the particular allergen. 106 monosensitized patients were included for each allergen source, except for mugwort and ragweed. Cross-reactivity was common in patients sensitized to mugwort and ragweed using extracts due to shared allergens; therefore, 25% of the 106 patients included in each of these two groups were serologically doublesensitized.

| Analysis 4. Specificity of major marker allergens
2 and six additional subjects). As mentioned above, prick tests to 13 aeroallergens and sIgE to seven extracts in the ImmunoCAP were negative.

| Skin tests
Skin prick tests were performed with extracts from ALK-Abelló, Hørsholm, Denmark. Test results were considered positive in a wheal larger than 3 mm in diameter and erythema.

| Singleplex sIgE testing
Specific IgE antibody levels in the patients' sera were measured using the ImmunoCAP 1000 platform. Specific IgE levels were expressed in kilo units per litre (kU/l), and sIgE values ≥0.35 kU/l were considered positive. For statistical analysis, levels >100 kU/l were rated as 100 kU/l.

| Multiplex sIgE testing
The multiplex test system ALEX 2 was performed according to the manufacturer's instructions. Specific IgE levels were expressed in kU/l, and sIgE values ≥0.30 kU/l were considered positive.

| Sample size calculation & statistical analyses
Sample size calculation was performed using the statistical software

| RESULTS
Demographical and clinical description of the four study populations (analysis 1: prevalence of (multiple) pollen sensitizations; analysis 2: sensitization to CCDs, profilins, and polcalcins; analysis 3: sensitivity of MAD; analysis 4: specificity of MAD) are shown in Table 1.

| Prevalence of pollen sensitization
1660 (56.3%) of the 2948 patients were extract sIgE positive to at least one of the six seasonal allergen sources investigated, and 1627 (55.2%) were positive to at least one of the five pollen allergens.
Multiple sensitizations to pollen were found in 34.0% of the overall study population. In the pollen-sensitized patients, sensitization to all five pollens was found in 22.4%; 6.7% had four sensitizations, 12.8% had three, 19.8% had two, and 38.4% of the patients were positive to only one pollen source. Many sensitizations were associated with low levels of sIgE (16.9% of all positive pollen sIgE results in analysis 1 were below 0.7 kU/l), and considering cut-offs above 0.7 or 3.5 kU/l, only 15.4% or 4.5% of the patients were positive to all five pollen species investigated (compared to 22.4% without cut-off).

| Correlation of sIgE and prick test results
Specific IgE and prick test results of the 2948 patients are shown in Table 2. It was remarkable that sIgE negative/prick positive patients were rarely seen for all allergens, but sIgE positive/prick negative patients were frequently observed for pollen allergens but not for F I G U R E 1 Flow-chart of the four analyses performed. In total, clinical data and test results of 3.590 patients were investigated. Patients of analysis 2 were randomly selected from analysis 1 (6 � 100 patients with 0-5 pollen sensitizations). The control group from analysis 2 (100 patients without pollen allergy) and six additional patients were used for analysis 4.
Alternaria. On average, 13.3% of the 14740 pollen extract determinations had negative corresponding prick test results, compared to 2.9% of the Alternaria extract determinations. The difference of 10.4% may be explained by anti-CCD IgE, reacting with pollen but not with Alternaria extract.

| Sensitizations to CCDs and pollen panallergens
Patients were grouped according to their number of pollen sensitizations (0-5), with one hundred randomly selected patients in each group. In these 600 sera (500 from patients with and 100 from controls without pollen allergy), MUXF3, Phl p 7, and Phl p 12 as markers for CCDs, polcalcins, and profilins, respectively, were determined ( Table 3). As expected, sensitizations to CCDs and panallergens were most frequently observed in patients with positive results to all five pollen species; in this group, 73% were sensitized to CCDs or any panallergen, and 17% even showed sIgE to two panallergens or one panallergen and CCDs. In the group with four positive pollen extracts, pan-sensitization was still relevant and observed in 17% of the patients, whereas this was rarely seen from group three sensitizations downwards. None of the 500 pollen allergic patients showed sIgE to all, CCDs and both panallergens.
After extrapolating from 500 to 1627 patients with pollen sensitizations, up to 18.5% could react to at least one panallergen or CCDs.
Specifically, sIgE to CCDs, profilins, and polcalcins could be expected in 8.7%, 10.9%, and 2.9%. None of the 100 patients in the control group did show any reactivity to CCDs, profilins, or polcalcins. After extrapolating from 600 to 2948 patients, up to 10.2% of all patients referred to our outpatient clinic for allergy diagnostics due to respiratory symptoms could be sensitized to at least one panallergen or CCDs (in detail, 4.8% to CCDs, 6.0% to profilins, and 1.6% to polcalcins).
We did not observe any sensitization to profilins or polcalcins without concomitant reactivity to at least one major pollen allergen in any of the 600 investigated sera. The primary mono-sensitizer in sera with profilin or polcalcin sensitization was timothy grass, birch, or ash pollen in 18.2%, 3%, and 3%, respectively, whereas multiple genuine pollen sensitizations were detected in 75.8%. Moreover, only T A B L E 1 Demographic data of the study populations. Asthma and bronchitis are defined by patients reporting chronic cough with and without an established diagnosis of asthma by a lung specialist.

| Correlation of sIgE to extracts and major molecular marker allergens
In

| Sensitivity of MAD
The sensitivity of major molecular allergens was investigated in 106 genuinely sensitized subjects per allergen source. Sensitivity was high, with percentages between 91.5% and 100% using ALEX 2 and 96.2% and 100% using ImmunoCAP. In addition, the molecular sensitivity correlated to pollen extract sIgE levels and increased with higher sIgE to the extracts (

| Specificity of MAD
The major molecular allergens Alt a 1, Amb a 1, Art v 1, Bet v 1, Fra/ Ole e 1 and Phl p 1 were tested in 106 non-allergic controls. None of these 106 subjects was positive for any of these molecular marker allergens resulting in a 100% specificity for all allergens. T A B L E 3 Reactivity to CCDs and the panallergens profilin and polcalcin. Patients were put into groups 0-5 according to the number of positive pollen extracts (ash, birch, timothy grass, mugwort, ragweed) with n = 100 for each group. In the control group (0), all pollen extracts were negative. Pan-sensitizations: Coincidence of CCDs, profilin, and polcalcin sensitization. Total percentages were extrapolated for the overall study population (n = 2948).

Number of positive pollen extracts CCD (MUXF3) Profilin (Phl p 12) Polcalcin (Phl p 7)
Pan-sensitizations    Conversely, if only three or fewer out of these five pollens were positive, the risk of cross-reactivity was minimal (5%) or negligible (≤1%). This approach helped to recognize cross-reactivity but still requires careful interpretation of the test results.
The second approach to minimize the risk of cross-reactivity is molecular allergology. In a recent review article by Barber et al, the benefit of molecular allergology and its impact on specific allergy diagnosis and therapy has been shown. 10 In addition, multiple studies have demonstrated that the use of molecular allergology leads to different therapeutic decisions compared to extract-based testing. [11][12][13] The reported sensitivities of molecular pollen marker allergens are promising;    (Table 4). It is known that the sensitivity of multiplex systems, such as ALEX 2 , could be lower in patients with low sIgE levels due to higher limits of detection, higher coefficients of variation, and potential inhibition by antigen-specific IgG. 42 We previously showed that the higher the sIgE to allergen extracts, the better was the sensitivity of molecular allergy testing in diagnosing house dust mite allergy. 43 The high rate of patients with low sIgE to mugwort may explain the observed lower sensitivity of Art v 1. However, because its sensitivity was equal to the extract-based approach in patients with sIgE levels greater than 0.7 kU/l in both methods and because the major allergen content of mugwort allergen immunotherapies is adjusted to Art v 1, it is definitely suitable for diagnostic use.
The singleplex assay ImmunoCAP and the multiplex platform ALEX 2 correlated strongly and produced almost the same results when the diagnosis was performed with major molecular allergens.
Both methods are appropriate for routine diagnostics though neither system is perfect: drawbacks of the ImmunoCAP are the costs for a comprehensive MAD and, theoretically, interference of anti-CCD antibodies with the cellulose used as a solid-phase allergen carrier. 44 On the other hand, multiplex platforms such as ALEX 2 may have a lower sensitivity in patients with very low levels of sIgE. However, a broad application of MAD in pollenallergic patients with any method could enhance the quality of allergy diagnosis and, consequently, the effectiveness of allergen immunotherapy.
The main limitation of our study, besides its retrospective design, is that we have investigated a Central European study population.
Therefore, additional allergens (e.g. Phl p 2 or 4) or allergen sources (e.g. olive tree, parietaria or plantain pollen) may be relevant in other distinct geographical regions, and the sensitization rates to CCDs or pan-allergens may vary.
In summary, about one-fifth (18.5%) of our pollen-sensitized patients and about one-tenth (10.2%) of all our patients referred to allergy diagnosis were affected by cross reactivity; therefore, crossreactivity is common and may lead to misdiagnosis and consequently to a 3-year immunotherapy with inadequate allergen vaccines. In contrast, MAD determining Alt a 1, Amb a 1, Art v 1, Bet v 1, Fra/Ole e 1 and Phl p 1 with commercially available methods is highly sensitive and specific and helps to avoid misdiagnosis and select primary allergen sources for immunotherapy.